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. 2020 Feb 4;14:46.
doi: 10.3389/fnins.2020.00046. eCollection 2020.

Acute Treatment With Gleevec Does Not Promote Early Vascular Recovery Following Intracerebral Hemorrhage in Adult Male Rats

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Free PMC article

Acute Treatment With Gleevec Does Not Promote Early Vascular Recovery Following Intracerebral Hemorrhage in Adult Male Rats

Mohammed Abbas et al. Front Neurosci. .
Free PMC article

Abstract

Intracerebral hemorrhage (ICH) remains one of the most debilitating types of stroke and is characterized by a sudden bleeding from a ruptured blood vessel. ICH often results in high mortality and in survivors, permanent disability. Most studies have focused on neuroprotective strategies designed to minimize secondary consequences and prevent further pathology. Lacking is an understanding of how ICH acutely affects cerebrovascular components and their response to therapeutic interventions. We hypothesized that ICH alters cortical vessel complexity in the parenchyma adjacent to site of the initial vascular disruption and that vascular abnormalities would be mitigated by administration of the PDGFR inhibitor, Imatinib mesylate (Gleevec). Briefly, ICH was induced in male adult rats by injection of collagenase into basal ganglia, followed by Gleevec administration (60 mg/kg) 1 h after injury. Rats were then perfused using vessel painting methodology (Salehi et al., 2018b) to stain whole brain vascular networks at 1 day post-ICH. Axial and coronal wide field fluorescence microscopy was performed. Analyses for vascular features were undertaken and fractal analysis for vascular complexity. Data were collected from four groups of rats: Sham + Vehicle; Sham + Gleevec; ICH + Vehicle; ICH + Gleevec. Microscopy revealed that cortical vessels in both ipsi- and contralateral hemispheres exhibited significantly reduced density and branching by 22 and 34%, respectively. Fractal measures confirmed reduced complexity as well. Gleevec treatment further reduced vascular parameters, including reductions in vessel density in tissues adjacent to the ICH. The reductions in brain wide vascular networks after Gleevec in the current study after ICH is contrasted by previous reports of improved behavioral outcomes and decreased lCH lesion volumes Reductions in the vascular network after Gleevec may be involved in long-term repair mechanisms by pruning injured vessels to ultimately promote new vessel growth.

Keywords: cerebrovasculature; edema; fractal; magnetic resonance imaging; vessel painting; vessels.

Figures

FIGURE 1
FIGURE 1
Intracerebral hemorrhage (ICH) results in cerebral vascular decrements at 24 h after injury or treatment. (A) Axial: The top row illustrates representative axial vessel painted whole brains in ICH animals with and without Gleevec treatment (* = injection site). The middle row are sample classical vascular analysis maps where red denotes vessels and blue demarcates vessel junctions. The third row displays fractal maps utilized for analysis of vascular complexity. (B) Coronal: Classical vascular analysis of coronal sections from all experimental groups wherein the ICH lesion in the striatum can be visualized in the injured rats (*). Middle row are the corresponding fractal analysis maps. The last row of coronal images are from susceptibility weighted magnetic resonance imaging (SWI MRI) which was utilized to monitor the extent of the hematoma and edema after ICH induction. Sham animals exhibited minimal vascular alterations from needle insertion whereas ICH animals have prominent and extensive areas of vessel disruption at the site of the ICH. All images in each column are from the same animal.
FIGURE 2
FIGURE 2
ICH induces modification of vascular features at 24 h post-injury. Axial Cortex: Classical vascular analysis of the entire axial cortex, ipsilateral (Ipsi) and contralateral (Contra) cortical hemispheres resulted in significant reductions in vessel density (A) and branching index (B) after ICH compared to shams. There was a concomitant increase in lacunarity (C) but not in the contralateral hemisphere. Fractal derived vascular complexity of the ipsilateral cortex using local fractal dimension histograms (LFD) (D) exhibited significant decrements in skewness and kurtosis between shams and ICH animals. Coronal Cortex and Basal Ganglia: Classical vascular analysis of coronal vessel painted sections at the level of the ICH lesion revealed significant reductions in vessel density (E) and reduced branching indices (F). There was a dramatic increase in lacunarity (G). Quantitative analysis of the LFD histograms from the ipsilateral cortex and basal ganglia exhibited no significant changes in skewness, kurtosis or peak frequency (H) in ICH vehicle compared to sham vehicle animals (t-test **p < 0.03, *p < 0.05).
FIGURE 3
FIGURE 3
Gleevec treatment does not improve ICH parenchymal vascular measures. Axial Cortex: Ipsilateral cortical vascular analysis revealed no significant changes in vessel density (A) or branching index (B) between Vehicle nor ICH Gleevec treated rats. However, significant reductions in vessel density were observed between sham vehicle and ICH + Vehicle and ICH + Gleevec rats. Vascular branching was significantly reduced between Sham + Vehicle and ICH + Gleevec rats. Lacunarity (C) was only increased in all groups relative to sham vehicle treated rats. Quantitative analysis of the distribution of LFD histograms (D) exhibited no significant reductions in skewness, and peak frequency in sham + Gleevec compared to sham + Vehicle animals. Kurtosis was significantly reduced between sham groups. Coronal Cortex and Basal Ganglia: Classical vascular analysis of ipsilateral cortex and basal ganglia revealed a progressively significant decrement in vessel density (E) across all groups. Post-hoc testing found significant differences between Sham + Vehicle and ICH groups (Vehicle, Gleevec). Branching index (F) and lacunarity (G) in sham nor ICH animals were not altered, except for a significant increase in lacunarity between sham + Vehicle and ICH + Vehicle rats. The distribution of LFD histograms (H) exhibited significant decreases in skewness, kurtosis and peak frequency across groups (p < 0.05) but post-hoc testing observed significant decreases only in sham groups. (one way Anova with Tukey’s multiple comparisons, **p < 0.03, *p < 0.05).
FIGURE 4
FIGURE 4
Gleevec treatment does not improve the morphology of the cortical vasculature. (A) Vessel painted brains from the axial surface were imaged using confocal microscopy and then subjected to classical vascular analysis. ICH vehicle and ICH Gleevec animals exhibited uniform staining of large, intermediate and small vessels within the cortex. Note the apparent loss of fine branching vessels in the ICH + Gleevec rats. (B) In coronal sections from vessel painted brains, the cortical vessels descending into the cortex exhibited a dense plexus of vessels in ICH Vehicle treated rats. In contrast, ICH Gleevec treated rats showed a reduced vascular plexus with fragmented vessels and regions with overt loss of vasculature (see Figure 5 for quantification).
FIGURE 5
FIGURE 5
Confocal vascular analysis also demonstrated that Gleevec treatment does not improve vascular density. (A) Quantitative assessment of axial cortical vascular features adjacent to the ICH injection site, revealed significant decrements in vascular density and trending decreases in vessel branching after Gleevec treatment. (B) Vascular features from cortical sections from coronal tissues also showed decreased vascular density and branching but did not reach significance. Note that both cortical assessments (axial vs. coronal) had similar relative decreases in vascular features (see Figure 4 for images) (*p < 0.05).
FIGURE 6
FIGURE 6
Confocal analysis of the basal ganglia vasculature revealed no improvement with Gleevec treatment. (A) The basal ganglia vasculature was assessed for morphological characteristics adjacent to the ICH site. As in the cortex, there was reduced vascular density with loss of fine vascular structures, including branch points. Far right pane illustrates the analysis that derived vessel density (red) and branching (blue dots). (B) Quantification of vascular metrics confirmed the visual observations with significantly reduced vascular density (p = 0.05) and trending decreases in (C) vessel branching (p = 0.09) in Gleevec treated compared to Vehicle treated rats.
FIGURE 7
FIGURE 7
Lesion and hemorrhage volumes were not modified by Gleevec treatment 24 h after ICH. (A) Representative MR images from Vehicle and Gleevec treatment rats. Shams exhibited cortical edema (white arrow) at the site of needle insertion. In ICH animals there were hypo- and hyperintense regions visible in the basal ganglia, consistent with blood (red dotted line) and edema (yellow dotted line), respectively (white arrows). (B) Subcortical hematoma volumes were not significantly altered between shams and Gleevec treated rats. (C) Volume of subcortical edema volumes was not significantly different following Gleevec treatment. (D) Correlations of total ICH lesion (blood + edema) volumes compared to ipsilateral fractal properties of ICH vehicle treated rat’s revealed significant correlations. The lesion volume was strongly correlated to fractal skewness (R2 = 0.6725, p = 0.0127), kurtosis (R2 = 0.6665, p = 0.0134), and peak frequency (R2 = 0.6290, p = 0.0189), but not for LFD (R2 = 0.1194, p = 0.4019). See Table 1 for additional correlations.

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